DNA Technology and Genomics Chapter 14 DNA Technology and Genomics

Overview: Understanding and Manipulating Genomes Sequencing of the human genome was largely completed by 2003 DNA sequencing has depended on advances in technology, starting with making recombinant DNA. Many are disappointed cures have not been found. What organisms have been sequenced? Look here: http://www.genomenewsnetwork.org/resources/sequenced_genomes/genome_guide_p1a.shtml

LE 20-2 Bacterium Cell containing gene of interest Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed by gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

One possible combination Recombinant DNA molecule Restriction site DNA 5¢ 3¢ 3¢ 5¢ Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme One possible combination DNA ligase seals the strands. Recombinant DNA molecule

LE 20-7 PCR – polymerase chain reaction. Making millions of copies of a portion of DNA to test. Thanks to bacteria from Yellowstone(with a heat stable DNA poly) – the reason forensics has evolved, sequencing the mammoth, Neanderthal etc.. We will do this on you!! 3 parts – heat DNA slightly to separate, cool so primers can bond, DNA poly adds nucleotides to 3” ends. Primers New nucleo- tides

LE 20-8 Mixture of DNA Longer molecules molecules of differ- Cathode ent sizes Longer molecules Cathode Shorter molecules Power source Gel Glass plates Anode

LE 20-9 Normal b-globin allele 175 bp 201 bp Large fragment Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Large fragment Ddel Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles RFLP – restriction length polymorphisms. Mutations that can be detected to see if you have disease causing alleles.

Future Directions in Genomics Genomics is the study of entire genomes. Same gene can make different proteins by splicing introns at different places!! How do we know what a gene does? Knock it out and see what the consequences are. Gene targeting is often used to inactivate single genes. Such gene 'knockout' experiments have elucidated the roles of numerous genes in embryonic development, adult physiology, aging and disease. To date, more than ten thousand mouse genes (approximately half of the genes in the mammalian genome) have been knocked out. Ongoing international efforts will make 'knockout mice' for all genes available within the near future.

Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes – like sickle cell. Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of good genes into cells – like viruses which can easily fit receptors. Retroviruses make a DNA copy with the gene Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

LE 20-16 Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow Inject engineered cells into patient.

Animal Husbandry and “Pharm” Animals Transgenic organisms are made by introducing genes from one species into the genome of another organism Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles)

Several organisms have similar genes that can be transferred between them. Ex: homeotic genes

Genetic Engineering in Plants Agricultural scientists have endowed a number of crop plants with genes for desirable traits The Ti plasmid is the most commonly used vector for introducing new genes into plant cells. Plants can produce a toxin from a bacteria, so when a caterpillar eats the leaf – its stomach explodes. Plants can also be “Round-up” resistant, so the weeds die from it but not the plants. Many plants world wide are now GMO’s – golden rice (w/ vitamin A for blindness) strawberries, tomatoes, corn etc…

LE 20-19 Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid Plant with new trait

Cloning – entire organism. A way to bring back or prevent extinctions? Take DNA from a host cell (skin, hair) suck out DNA from an egg and put new DNA in. Implant in a surrogate mother (must be related enough to not reject embryo). Wait until birth. Dolly and many mammals have been cloned. Problems with disease and aging due to methylation of older DNA?? Genes not turning on as they do in infancy. Conclusion – clone in mammals not identical, not same personality

SNP – single nucleotide polymorphism Spot where 1 base pair differs in at least 1% of the population. It’s a stable mutation so it helps track ancestry.